Although clathrin-mediated endocytosis (CME) is by far the best understood pathway for entry into the cell, much remains to be learned regarding how components of the endocytic machinery work coordinately to sort cargo, deform the membrane and pinch off clathrin coated vesicles (CCVs). CME functions in nutrient uptake, to regulate the expression of surface receptors and transporters and to control the signaling activity of receptor tyrosine kinases and GPCRs. At the synapse CME is highly regulated in both space and time to accomplish rapid and efficient recycling of synaptic vesicles during neurotransmission. Long considered a constitutive process, we now appreciate that CME in nonneuronal cells is also highly regulated. In collaboration with Gaudenz Danuser, my lab has applied quantitative live cell total internal reflection fluorescence microscopy to analyze the dynamic behaviors of clathrin coated pit (CCPs) and to quantify rates of initiation, maturation and the release of CCVs. These studies have revealed that a large fraction of nascent CCPs fail to mature and are aborted. This and other findings over the past 8 years, have led us to propose the existence of an endocytic checkpoint that serves as a fidelity monitor for early CCP assembly. Key regulatory events that govern transition through this checkpoint occur at early stages following nucleation of nascent CCPs. We recently demonstrated that the recruitment of endocytic accessory proteins (EAPs) through interactions with the adaptor protein complex, AP2, as well as the early recruitment of dynamin-2 to nascent CCPs is critical determinants of CCP maturation. We also recently discovered that nonneuronal cells with impaired CCP maturation, enlist dynamin-1 to bypass the endocytic checkpoint and accelerate CME. Aims 1 and 2 of this proposal will dissect and computationally model the roles of AP2 complexes and dynamin, respectively, in these critical early events to elucidate novel mechanisms that ensure rapid and efficient CME. While many components of the endocytic machinery are known, and have been grouped into modules based on the temporal hierarchy of their recruitment to CCPs. Few EAPs have been functionally characterized and whether these temporal modules reflect functional interactions remains unknown. Potential redundant or synergistic functional interactions can be identified through genetic interactions and while several siRNA based screens have been conducted to identify components of the endocytic machinery, remarkably, these have failed to identify even known EAPs, potentially due to the inability of previous screens to detect partial effects, functional redundancy and/or, as we recently discovered, the robustness and plasticity of CME. In Aim 3, we propose a new approach to screen for defects in CME and early endosomal trafficking and to conduct a massively parallel, genome-wide shRNA screen and follow-up epistasis mapping to identify functional relationships between components of the endocytic machinery. Together these studies will provide unprecedented new insight into factors that determine CME efficiency and robustness.